The objective of this collaborative program is to develop a novel microchip sensing system capable of sensitively, selectively, simultaneously and rapidly identifying the presence of biowarfare (BW) agents relevant to our nation's biodefense program (NIAID Category A, B & C priority pathogens). Such development will meet the urgent need for cost-effective biodefense diagnostics for both public health laboratories and point-of-care use to identify or diagnose individuals exposed to agents and toxins of category A-C pathogens. The resulting microchip device will ultimately be applicable to agent detection in or on symptomatic or exposed individuals, in addition to preventative monitoring of food, air and water for general health safety, and is, thus, expected to dramatically change the way biodetection and diagnostic assays are performed. Most importantly, the proposed microchip device will be capable of performing an entire immunoassay on several pathogens of interest, simultaneously and in a single step on the microchip. This joint, interdisciplinary effort will combine fundamental and practical studies for creating a miniaturized (self-contained), field-deployable biowarfare (BW) agent analyzer based on the coupling of immunoassays, rapid electrophoretic separations, and chemiluminescence/electrochemical detections on a chip platform. This new, completely functional microlaboratory will integrate multiple/parallel assays for different target pathogens or toxins, along with the necessary sample manipulations onto a single microchip platform. Antibodies to selected BW agents will be enzyme-labeled and purified. The selected agents include: Staphylococcus enterotoxin B, ricin toxin, botulinum toxin, Epsilon toxin of Clostridium perfringens, E. coli toxin, Brucella abortus, Bacillus anthracis, Francisella tularensis and Salmonella. Enzymatic amplification of the signal will be provided by conjugating the antibodies to one of four enzyme labels: horseradish peroxidase, alkaline phosphatase, glucose oxidase and beta-galactosidase. The pre-column immunological reaction will be followed by electrophoretic separation of the enzyme-labeled antibody from the enzyme-labeled antibody-antigen pair. Finally, on-chip post-column introduction of an appropriate substrate will enable the sensitive detection of the antigen by either chemiluminescence or amperometric means. The entire assay will take place in a single step on the microchip. Preliminary results in this direction (including extremely low detection limits) are very encouraging. The successful utility of this new microsystem will depend upon a complete understanding of its fundamental behavior. The proposed research aims at gaining such insights into the on-chip bioassays, and for using this new knowledge for the rationale development of a new, micromachined BW agent analyzer. The primary goal is to understand the impact of the enzyme labeling upon the recognition capabilities of selected antibodies, learn how immunological reactions behave on a small scale and how they can be interfaced with separation microchips, and discover how the microfluidics can be tailored to suit the requirements of particular BW agent immunoassays.